Maintenance of healthy bone requires the coupled activities of osteoclasts (OCs), which resorb the organic and inorganic components of bone, and osteoblasts (OBs), cells responsive for production and calcification of matrix proteins. Both cell types execute their function by regulated exocytosis, a process in which specialized vesicles are targeted to specific cell domains, leading to polarized secretion. Thus, we hypothesize that secretory vesicle proteins of the OB are important for OB function and maintenance of bone mass. In contrast to recent insights into transcriptional regulation of osteoblast differentiation, the mechanisms by which osteoblasts secrete matrix proteins to form bone are largely unknown. As a Pilot and Feasibility study, our goal is to identify the molecular mechanisms by which secretory vesicle proteins regulate osteoblast function. In this regard, we have developed experimental tools enabling us to 1) immunopurify lysosomes and thus determine their protein content by mass spectrometry, 2) knockdown endogenous candidate secretory genes using lenti- and retro- viral-mediated transduction of shRNA and rescue it with an RNAi-resistant mutant. We are thus in a position to a) identify novel secretory vesicle proteins in OBs, and b) test their role in secretion and bone formation in vitro. While these proposed studies will identify a number of candidate regulatory proteins in OBs, one, osteoactivin (OA) is presently in hand. OA is expressed in OCs and OBs, localizes in lysosomes in both cell types and regulates bone formation and resorption, in vitro. Moreover, a murine model of OA mutation is already available. Thus, our specific aim is A) to identify osteoblast secretory vesicle proteins by mass-spectrometry and to characterize their bone forming functions by shRNA-mediated gene knockdown in vitro, and B) in parallel, to characterize the role of the candidate OB secretory protein OA in bone formation in vivo using an existing murine model. The latter study will be done with assistance from two core facilities in the Washington University Core center for Musculoskeletal Biology and Medicine, namely Core B-Musculoskeletal Structure and Strength, and Core C-ln situ Molecular Analysis, Following completion of these studies, we will be in a position to propose in vivo functional studies of additional candidate genes identified by our OB proteomics analysis as part of a future R01 proposal.